The present invention concerns a pharmaceutical composition, its use in tumour diagnosis, therapy and prevention, methods for diagnosing, treating and preventing tumours as well as antibodies and their use.
The unregulated growth of tumour cells is caused by a new expression pattern of genes which regulate the cell cycle control, adhesion, angiogenesis, invasiveness and finally the formation of metastases (Pardee, Advances in Cancer Res. 65 (1994), 213-227; Ponta et al., Biochem. Biophys. Acta 1198 (1994), 1-10). The clinical course of tumour diseases such as breast cancer is characterized by several defined molecular processes such as oestrogen-independent growth, tamoxifen resistance, expression of vimentin, increase in invasiveness and finally cross-resistance towards a large number of chemotherapeutic agents which is often referred to as multi-drug resistance (cf. e.g. Clarke et al., J. Endocrinol. 122 (1989), 331-340; Sommers et al., Cancer Res. 53 (1992), 5190-5197; Sommers et al., Cancer Res. 49 (1989), 4258-4263 and Saceda et al., Mol. Endocrinol. 2 (1988), 1157-1162).
A key concern of tumour research is to identify genes which play an important role in the progression of tumour diseases and to provide new agents for tumour diagnosis, prevention or therapy on the basis of these genes.
The present invention describes the identification, cloning and characterization of the gene for a polypeptide which is named progression-associated protein xe2x80x9cPAPxe2x80x9d. This protein is expressed in the metastasising human mammary carcinoma cell line MCF-7ADR whereas no expression was found in the non-metastasising mammary carcinoma cell line MCF-7. Hence the PAP protein, a nucleic acid coding therefor as well as antibodies directed against the protein are suitable as agents for the diagnosis, therapy or prevention of tumour diseases and especially of breast cancer.
The invention provides a pharmaceutical composition which is characterised in that it contains the following active components:
(A) a nucleic acid which comprises
(a) the protein-coding nucleotide sequence shown in SEQ ID NO.1,
(b) a nucleotide sequence corresponding to the sequence from (a) within the scope of the degeneracy of the genetic code,
(c) a sequence hybridizing with the sequences from (a) or/and (b) under stringent conditions or
(d) an at least 20 nucleotide long section of the sequences from (a), (b) or/and (c),
(B) a polypeptide or peptide which is coded by a nucleic acid according to (A) or/and
(C) an antibody to a polypeptide or peptide according to (B).
In this connection stringent hybridization conditions are understood to mean that a hybridization still occurs after washing at 55xc2x0 C., preferably at 62xc2x0 C., particularly preferably at 68xc2x0 C. in a low salt buffer (e.g. 0.2xc3x97SSC, 0.1% SCS) (see also Sambrook et al., (1989), Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press).
In a preferred embodiment the pharmaceutical composition additionally contains common pharmaceutical carrier substances, auxiliary substances or additives.
The protein-coding nucleotide sequence shown in SEQ ID NO. 1 codes for a protein with a length of 157 amino acids which corresponds to the B4B nucleotide sequence described by Ruegg et al., 1996 (B4B, a Novel Growth-Arrest Gene, Is Expressed by a Subset of Progenitor/Pre-B Lymphocytes Negative for Cytoplasmic xcexc-Chain, Am.Ass.Imm., 1995, p. 72-80) and has a homology to already known proteins. In the experiments carried out by Ruegg et al., (supra) the expression of the B4B protein is claimed to arrest the growth of Cos-7 cells. Hence the use of the B4B protein is postulated as a potential tumour suppressor.
One protein which is homologous to PAP is the rabbit protein CL-20 which is induced in vitro during the differentiation of rabbit tracheal cells and occurs most frequently in squamous epithelial tissue (Marvin, J. Biol. Chem. 270 (1995), 28910-28916). The expression of CL-20 does not depend on the growth conditions but retinoids which inhibit the differentiation of squamous epitheleum repress the induction of CL-20.
A further protein which is homologous to PAP is the rat epithelial membrane protein EMP-1 which mainly occurs in the proliferation and differentiation zones of the outer gastric gland as well as in epithelial cells of the stomach region (Taylor et al., J. Biol. Chem. 270 (1995), 28824-28833).
PAP is also homologous to the peripheral myelin protein PMP22 from man, mouse and rat which is a myelin-associated transmembrane structural protein. PMP22 is a specific protein for growth inhibition which prevents cell cycle progression (Hayasaka et al., BBRC 186 (1992), 827-831; Edomi et al., Gene 126 (1993), 289-290; Welcher et al., Proc. Natl. Acad. Sci. USA 88 (1991), 7195-7198; Spreyer et al., EMBO J. 10 (1991), 3661-3668).
On the basis of the data of Ruegg et al. and the homology to the PMP22 protein responsible for maintaining the cellular dormant state, it was extremely surprising that the PAP protein is selectively expressed in the strongly degenerate metastisising cell line MCF-7ADR but not in the less degenerate cell line MCF-7.
Furthermore PAP has a high degree of homology to previously not yet published proteins whose sequences are stored in the gene library. These proteins are referred to as murine TMP or human TMP and have an amino acid identity of 76% or 95% to PAP.
The nucleotide and amino acid sequences of murine TMP are shown in SEQ ID NO. 3 and 4 and of human TMP are shown in SEQ ID NO.5 and 6. PAP and human TMP mainly differ in the region of the amino acids 32-48.
A further subject matter of the present invention is the use of a vector which contains at least one copy of a nucleic acid as previously defined or a section thereof for the transformation of a cell. This nucleic acid can for example be genomic DNA, cDNA or mRNA. It is preferably a recombinant DNA molecule.
The invention also concerns the use of a vector which contains an at least 20 nucleotide long section of the protein-coding sequence shown in SEQ ID NO.1. This section preferably contains a nucleotide sequence specific for PAP. These nucleic acids are especially suitable for preparing antisense nucleic acids that can be used therapeutically which preferably have a length of up to 50 nucleotides.
The vector containing the nucleic acid can be capable of replication in eukaryotes or prokaryotes. It can be a vector that can be integrated into the genome of the host cell e.g. bacteriophage xcex, or it can be a vector that is present extrachromosomally (e.g. a plasmid). The vector according to the invention can be obtained by subcloning the PAP-DNA into a base vector. Such base vectors and in particular vectors containing the elements necessary for protein expression are familiar to a person skilled in the art.
When cloning a nucleic acid coding for PAP, it is possible to prepare an expression vector that can be expressed in a suitable host cell to form the protein according to the invention. Preferred host cells are microorganisms such as E. coli or yeast and also higher cells such as mammalian or insect cells. Preferred expression vectors are for example plasmids, bacteriophage xcex for prokaryotes, yeast vectors or viral vectors for higher cells e.g. SV40, vaccinia, baculovirus. With regard to the expression of a PAP-coding nucleic acid reference is made in particular to the methods stated in Sambrook et al. (supra).
Systems suitable for expressing PAP contain suitable vectors e.g. the vector pcDNA1 (Invitrogen) in which the DNA to be expressed is under the control of the cytomegalovirus promoter.
A further subject matter of the present invention is a cell which is characterized in that it is transformed with a nucleic acid which comprises
(a) the protein coding nucleotide sequence shown in SEQ ID NO.1,
(b) a nucleotide sequence corresponding to the sequence from (a) within the scope of the degeneracy of the genetic code or
(c) a nucleotide sequence which has a homology of more than 95% to a sequence from (a) or/and (b) provided that the cell is not a Cos-7 cell.
The cell can be a eukaryotic as well as a prokaryotic cell. Methods for transforming cells with nucleic acids are part of the general state of the art and do not therefore need to be elucidated in more detail.
A further subject matter of the present invention is the use of a PAP polypeptide with the amino acid sequence shown in SEQ ID NO. 2, fragments of this polypeptide or variants thereof as an immunogen for producing antibodies.
Variants are understood as sequences which differ from the amino acid sequence shown in SEQ ID NO. 2 by substitution, deletion or/and insertion of individual amino acids or short amino acid sections.
The term xe2x80x9cvariantxe2x80x9d includes naturally occurring allelic variations of PAP as well as protein produced by recombinant DNA technology (in particular produced by in vitro mutagenesis with the aid of chemically-synthesized oligonucleotides) whose biological or/and immunological activity essentially corresponds to that of the protein shown in SEQ ID NO.2.
In a preferred embodiment the invention concerns the use of the pharmaceutical composition for tumour diagnosis and in particular for the diagnosis of mammary carcinomas. Furthermore an application as a tumour progression marker whose expression correlates with the progress of the tumour or/and with the degree of metastasis is especially preferred.
In a particularly preferred embodiment for the diagnosis of tumours the expression rate of the PAP protein correlates with the tumour stage and in particular with the tendency to form metastases.
A preferred embodiment is the use of the pharmaceutical composition for tumour therapy or prevention and an application for the therapeutic or preventive treatment of mammary carcinomas is particularly preferred.
Yet a further subject matter of the present invention is a method for diagnosing tumours in which a patient or a tissue derived from a patient is brought into contact with a pharmaceutical composition according to the invention and the expression of the PAP protein is determined qualitatively or/and quantitatively. This determination can for example be carried out at the nucleic acid level by using nucleic acid hybridization probes or by reverse transcription/PCR at the protein level by using antibodies in cytochemical or histochemical methods. The pharmaceutical composition is particularly preferably used as a tumour progression marker to identify the aggressiveness of tumours and especially of mammary carcinomas by quantifying the PAP expression for example after an operative procedure or to monitor the progress of a chemotherapeutic treatment.
Finally the present invention also concerns a method for the treatment or prevention of tumours in which a pharmaceutical composition according to the invention is administered to a patient which contains the active component in an amount that is effective against tumours. Specific examples of pharmaceutical compositions which are suitable for therapeutic purposes are for example bispecific antibodies and antibody-toxin or antibody-enzyme conjugates such as those used in the clinical studies for the HER-2 gene (cf. in this connection Weiner et al., Cancer Res. 55 (1995), 4586-4593; Weiner et al., J. Hematotherapy 4 (1995), 453-456; Repp et al., J. Hematotherapy 4 (1995), 415-421; Valone, J. Hematotherapy 4 (1995), 471-475; Rodrigues, Cancer Res. 55 (1995), 63-70).
A further subject matter of the invention is a method for treating tumours which is characterized in that the expression of the PAP protein in the tumour cells is reduced or eliminated.
The expression can be reduced by using agents or methods which interfere with transcription or/and translation or reduce the half-life of the mRNA.
For example anti-sense oligonucleotides to the nucleotide sequence shown in SEQ ID NO.1 can be introduced into the cells or the cell is transformed with an expression vector which expresses an anti-sense sequence to SEQ ID NO.1. Furthermore factors which interfere with the PAP mRNA expression or the PAP translation can be used.
The PAP expression can be completely switched off by using a so-called targeting vector for the PAP gene and carrying out a homologous recombination. The said methods are known to a person skilled in the art and therefore do not require further elucidation.
On the other hand it may be necessary for various purposes to increase the proliferation of cells if, for example, certain cell populations or subpopulations are required in large amounts or if it is intended to specifically stimulate the proliferation of certain cells in a tissue.
A further subject matter of the invention concerns a method for increasing the proliferation of a cell which is characterized in that
(A) a polypeptide or peptide is expressed in the cell or its expression is increased in the cell which is coded by
(a) a nucleotide sequence shown in SEQ ID NO.1,
(b) a nucleotide sequence corresponding to the nucleotide sequence from (a) within the scope of the degeneracy of the genetic code,
(c) a nucleotide sequence which has a homology of more than 95% to a nucleotide sequence from (a) or/and (b) or
(d) an at least 20 nucleotide long section of the sequences from (a), (b) or/and (c) or
(B) a polypeptide or peptide which is coded by a nucleic acid according to (A) is Introduced into the cell.
In addition to nucleic acids and polypeptides of PAP, antibodies to polypeptides or fragments thereof are also suitable as components of the pharmaceutical composition.
Polyclonal antibodies to a polypeptide as described above are particularly preferred provided that the antibodies are not directed towards the peptide sequence CSDSLSYASEDALK (SEQ ID NO. 7) or SHYANRDGTQYHH (SEQ ID NO. 8) or a monoclonal antibody to a polypeptide as described above is particularly preferred. An antibody is most preferred which is characterized in that it is directed towards a peptide sequence which corresponds to the amino acids 32 to 48, 49 to 62 or 119 to 129 of the amino acid sequence shown in SEQ ID NO.2.
In this connection antibodies can be produced in the usual manner by immunizing experimental animals with the complete PAP protein or fragments thereof and subsequently isolating the resulting polyclonal antisera. Monoclonal antibodies can be obtained in a known manner from the antibody-producing cells of the experimental animals by cell fusion according to the method of Kxc3x6hler and Milstein or further developments thereof. It is also possible to produce human monoclonal antibodies by known methods.
Peptides are preferred as the immunogen which are derived from the extracellular domains of PAP (cf. FIG. 2). Particularly preferred peptides are derived from regions which correspond to the amino acids 32-48, 49-62 or 119-129 of SEQ ID NO.2. These peptides are preferably coupled by known methods to a carrier e.g. keyhole limpet hemocyanin (Snipes et al., J. Cell. Biol. 117 (1992), 225-238). The resulting conjugates are used to immunize experimental animals e.g. rabbits.
Hence a further subject matter of the present invention is an antibody to the PAP protein or a variant thereof, preferably an antibody that exhibits no cross-reaction with homologous proteins such as EMP-1, PMP22 and CL-20. The antibody is particularly preferably directed towards a peptide sequence which corresponds to the amino acids 32 to 48, 49 to 62 or 119 to 129 of the amino acid sequence shown in SEQ ID NO.2.
A further subject matter of the invention is the use of the antibodies described above in an immunological method such as an immunoprecipitation, a Western blot, a competitive immunotest or a sandwich test.
Yet a further subject matter of the invention concerns the use of a polypeptide or a fragment thereof which is coded by
(a) the protein-coding nucleotide sequence shown in SEQ ID NO.1,
(b) a nucleotide sequence corresponding to the sequence from (a) within the scope of the degeneracy of the genetic code or
(c) a nucleotide sequence with a homology of more than 95% to a sequence from (a) or/and (b) to produce an antibody using a phage-display antibody library.
This method enables the identification and production of an antibody to the PAP protein only using methods of recombinant DNA technology without having to use animals or (primary) cells isolated from animals.
The method therefore does not require the tedious and time-consuming steps of a classical antibody production such as immunization of experimental animals, boosting or cell fusion and selection cycles for individual clones.
The provision of a pharmaceutical composition according to the invention, of PAP protein, of a nucleic acid coding therefor and of an antibody directed towards this protein are the prerequisites for a specific search for effectors of this protein. The target for these substances should be the potential extracellular domains of the protein which are located in the region of the amino acid residues 29 to 63 and 118 to 130 of the amino acid sequence shown in SEQ ID NO.2. Substances which inhibit or activate this region of the protein are able to selectively influence PAP-controlled cell functions. Hence they can be used to treat corresponding clinical pictures. In the case of clinical pictures which are due to a deficiency of PAP it would be possible to carry out a gene therapy comprising the transfer of a nucleic acid coding for PAP by means of vectors, e.g. viral vectors, into the appropriate target tissue. On the other hand in the case of clinical pictures which are due to an uncontrolled expression of PAP, a gene therapy can be carried out which leads to an inhibition of this expression.
Moreover, the results that have been presented also provide the basis for a specific diagnosis of diseases which are causally linked to changes in PAP activity. These investigations can be carried out with the aid of specific nucleic acid probes for the detection at the nucleic acid level i.e. at the gene or transcript level or with the aid of antibodies to PAP for detection at the protein level.